Bottom Line:
Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues.In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites.Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

ABSTRACTRegulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

fig4: αE-expressing subsets have a higher migratory capacity toward inflammatory chemokines. The chemotactic response of pooled spleen and lymph node T cells to 30 nM CCL19, 100 nM CXCL9, 100 nM CCL17, and 10 nM CCL20 was analyzed in an in vitro chemotaxis assay. The number of migrating cells of each subset was measured by flow cytometry. Results are expressed as the percentage of the indicated subset that migrated to the lower chamber and where normalized to the mean migration rate of all subsets. Shown is the mean ± SD of three (CCL17, CCL20) or four (CCL19, CXCL9) experiments (*, P < 0.05; **, P < 0.01). Basal migration toward medium alone (dotted lines) showed no significant differences between the subsets.

Mentions:
Chemokines have important functions in guiding distinct subsets of leukocytes into specific tissues or into inflamed areas. To confirm the results from the cDNA microarrays, where differential expression of several chemokine receptors between Treg subsets was observed, we performed quantitative RT-PCR (Table SI). In support of the microarray data, we observed an increased expression of CXCR3 mRNA on αE+CD25+ and especially on αE+CD25− cells compared with the αE−CD25+ subset. Additionally, αE single positive cells showed a slightly enhanced expression of CXCR4 mRNA compared with both CD25+ subsets. No significant differences were observed for CCR5 and CCR9 expression. In discordance to the microarray data, quantitative RT-PCR revealed slightly higher values for CCR7 mRNA expression in the αE+ subsets compared with the CD25 single positive cells, by unknown reasons. To get conclusive results, especially on functional properties, we performed chemotaxis assays, which also take into account that chemokine responsiveness often dissociates from receptor expression (26). For these assays, we took untouched total T cells freshly isolated from lymphoid organs and analyzed the responsiveness of the different subsets contained therein. All Treg subsets harbored high frequencies of cells responding toward the CCR7 ligand CCL19 (ELC), however, αE−CD25+ cells reproducibly showed a significantly higher migratory response toward CCL19 than both αE-expressing subsets in four experiments (Fig. 4). A contrasting pattern was found for the inflammatory chemokines CXCL9 (Mig), CCL17 (TARC), and also for CCL20 (LARC), which bind to the receptors CXCR3, CCR4, and CCR6, respectively. Both, αE+CD25+ and especially αE single positive cells displayed a significantly higher responsiveness toward these inflammatory chemokines than did CD25 single positive cells (Fig. 4). Titration of selected chemokines assured that the graduated response is not merely caused by different response curves (unpublished data). Furthermore, we tested CCL2 (MCP-1) and CCL4 (MIP-1β), which bind to the receptors CCR2 and CCR5, respectively. CCL2 and CCL4 did not induce a significant migration of freshly isolated cells at chemokine concentrations of 10 nM or 0.1–10 nM, respectively (unpublished data). In conclusion, αE+ cells respond preferentially to several inflammatory chemokines, whereas the CD25 single positive subset is more reactive to chemokines involved in recirculation through lymphoid tissues.

fig4: αE-expressing subsets have a higher migratory capacity toward inflammatory chemokines. The chemotactic response of pooled spleen and lymph node T cells to 30 nM CCL19, 100 nM CXCL9, 100 nM CCL17, and 10 nM CCL20 was analyzed in an in vitro chemotaxis assay. The number of migrating cells of each subset was measured by flow cytometry. Results are expressed as the percentage of the indicated subset that migrated to the lower chamber and where normalized to the mean migration rate of all subsets. Shown is the mean ± SD of three (CCL17, CCL20) or four (CCL19, CXCL9) experiments (*, P < 0.05; **, P < 0.01). Basal migration toward medium alone (dotted lines) showed no significant differences between the subsets.

Mentions:
Chemokines have important functions in guiding distinct subsets of leukocytes into specific tissues or into inflamed areas. To confirm the results from the cDNA microarrays, where differential expression of several chemokine receptors between Treg subsets was observed, we performed quantitative RT-PCR (Table SI). In support of the microarray data, we observed an increased expression of CXCR3 mRNA on αE+CD25+ and especially on αE+CD25− cells compared with the αE−CD25+ subset. Additionally, αE single positive cells showed a slightly enhanced expression of CXCR4 mRNA compared with both CD25+ subsets. No significant differences were observed for CCR5 and CCR9 expression. In discordance to the microarray data, quantitative RT-PCR revealed slightly higher values for CCR7 mRNA expression in the αE+ subsets compared with the CD25 single positive cells, by unknown reasons. To get conclusive results, especially on functional properties, we performed chemotaxis assays, which also take into account that chemokine responsiveness often dissociates from receptor expression (26). For these assays, we took untouched total T cells freshly isolated from lymphoid organs and analyzed the responsiveness of the different subsets contained therein. All Treg subsets harbored high frequencies of cells responding toward the CCR7 ligand CCL19 (ELC), however, αE−CD25+ cells reproducibly showed a significantly higher migratory response toward CCL19 than both αE-expressing subsets in four experiments (Fig. 4). A contrasting pattern was found for the inflammatory chemokines CXCL9 (Mig), CCL17 (TARC), and also for CCL20 (LARC), which bind to the receptors CXCR3, CCR4, and CCR6, respectively. Both, αE+CD25+ and especially αE single positive cells displayed a significantly higher responsiveness toward these inflammatory chemokines than did CD25 single positive cells (Fig. 4). Titration of selected chemokines assured that the graduated response is not merely caused by different response curves (unpublished data). Furthermore, we tested CCL2 (MCP-1) and CCL4 (MIP-1β), which bind to the receptors CCR2 and CCR5, respectively. CCL2 and CCL4 did not induce a significant migration of freshly isolated cells at chemokine concentrations of 10 nM or 0.1–10 nM, respectively (unpublished data). In conclusion, αE+ cells respond preferentially to several inflammatory chemokines, whereas the CD25 single positive subset is more reactive to chemokines involved in recirculation through lymphoid tissues.

Bottom Line:
Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues.In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites.Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

ABSTRACTRegulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.